Nanofluidic devices for the rapid mapping of whole genomes and related systems and methods of analysis

ABSTRACT

Devices and methods generate an ordered restriction map of genomic DNA extracted from whole cells. The devices have a fluidic microchannel that merges into a reaction nanochannel that merges into a detection nanochannel at an interface where the nanochannel diameter decreases in size by between 50% to 99%. Intact molecules of DNA are transported to the reaction nanochannel and then fragmented in the reaction nanochannel using restriction endonuclease enzymes. The reaction nanochannel is sized and configured so that the fragments stay in an original order until they are injected into the detection nanochannel. Signal at one or more locations along the detection nanochannel is detected to map fragments in the order they occur along a long DNA molecule.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/926,595, filed Oct. 29, 2015, which is a divisionalapplication of U.S. patent application Ser. No. 14/204,211, filed Mar.11, 2014, now U.S. Pat. No. 9,255,288, issued Feb. 9, 2016, which claimsthe benefit of and priority to U.S. Provisional Application Ser. No.61/778,746, filed Mar. 13, 2013, the contents of which are herebyincorporated by reference as if recited in full herein.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under Grant No. HG002647awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates to the genomic characterization of polynucleicacids.

BACKGROUND OF THE INVENTION

There has been considerable recent interest in the incorporation ofnanoscale components in lab-on-a-chip fluidic devices. This interestowes its origin to several advantages (and differences that may beadvantageously leveraged) in moving from the micron scale to thenanoscale. These differences include, for example, double-layer overlap(DLO) and its effect on electro-osmosis and charge permselectivity,localized enhancement of electric fields, higher surface to volumeratios, confinement effects on large synthetic and bio-polymers, and theemerging importance of entropic effects. See, e.g., Yuan et al.,Electrophoresis 2007, 28, 595-610; Schoch et al., Rev. Mod. Phys. 2008,80, 839-883; and Kovarik et al., Anal. Chem. 2009, 81, 7133-7140.Historic examples of nanoscale devices include the use of porous mediaand gels in chromatographic separations and filtration membranes withnanoscale pores. See, e.g., Lerman et al., Biopolymers 1982, 21,995-997; and Tong et al., M. Nano Lett. 2004, 4, 283-287. Recentefforts, however, have been focused on engineering geometricallywell-defined conduits for fluid and analyte transport and seamlesslyintegrating them into devices. See, e.g., Volkmuth et al., Nature 1992,358, 600-602; and Striemer et al., Nature 2007, 445, 749-753. Theadvantage of such regular structures is the relative simplicity ofpressure and field gradients, fluid flow, and molecular motion containedwithin, in contrast to these properties in more tortuous networks. Thecapability to define, characterize, and easily model these systems canallow a better understanding of separation mechanisms and singlemolecule physics, for example. See, e.g., Volkmuth et al., Nature 1992,358, 600-602; Reisner et al., Phys. Rev. Lett. 2005, 94, 196101; andSalieb-Beugelaar et al., Lab Chip 2009, 9, 2508-2523.

Recently FIB milling techniques have been described to form nanofluidicdevices. See, Menard et al., Fabrication of Sub-5 nm Nanochannels inInsulating Substrates Using Focused Ion Beam Milling, Nano Lett. 2011,11, 512-517 (published Dec. 20, 2010); and U.S. Provisional PatentApplication Ser. No. 61/384,738, filed Sep. 21, 2010 (and related PCTApplication PCT/US2011/052127), entitled, Methods, Systems And DevicesFor Forming Nanochannels, the contents of which are hereby incorporatedby reference as if recited in full herein. In addition to FIB milling, avariety of other methods suitable for nanochannel fabrication can beused, including, for example, electron beam lithography, nanoimprintlithography, photolithography, templating or molding strategies, andother methods understood by one of ordinary skill in the art.

A number of nanofluidic devices have been proposed, including those withintegrated miniature electrodes (nano- or micro-scale) forsingle-molecule sensing and/or nucleic acid sequencing. Alternatively,integrated devices consisting of entirely fluidic components can providegreater control of single-molecule transport and detection. See, Menardet al., A Device for Performing Lateral Conductance Measurements onIndividual Double-Stranded DNA Molecules, ACS Nano 2012, 12, 9087-9094(published Sep. 5, 2012); U.S. Provisional Patent Ser. No. 61/533,523,filed Sep. 12, 2011 (and corresponding pending PCT/US13/054128),entitled, Devices with a Fluid Transport Nanochannel Intersected by aFluid Sensing Nanochannel and Related Methods; and U.S. ProvisionalPatent Ser. No. 61/770,586, filed Feb. 28, 2013, entitled NanofluidicDevices with Integrated Components for the Controlled Capture, Trapping,and Transport of Macromolecules and Related Methods of Analysis, thecontents of which are hereby incorporated by reference as if recited infull herein. Such integration of components on a single monolithicdevice can enable new methods and systems that address current analysisneeds in fields such as DNA sequencing and medical diagnostics.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the invention are configured to provide devices thatfacilitate high throughput restriction mapping of chromosomal DNA.

Embodiments of the invention include nanofluidic analysis systems. Thesystems include: (a) a reaction nanochannel that is between 500 μm and10 cm long and that merges into a detection nanochannel at an interfaceposition therebetween where the detection nanochannel reduces in sizerelative to the reaction nanochannel; (b) a microfluidic channel incommunication with an ingress portion of the reaction nanochannel; (c) afirst electrode in communication with the microfluidic channel; (d) afirst transverse fluidic channel extending from and in fluidcommunication with the reaction nanochannel at a location that is spacedapart from but proximate the ingress portion of the reactionnanochannel; (e) a second electrode in communication with the firsttransverse fluidic channel; (f) a second transverse fluidic channelextending from and in fluid communication with the reaction nanochanneldownstream of the first transverse fluidic channel; (g) a thirdelectrode in communication with the second transverse fluidic channel;(h) a fourth electrode in communication with the detection nanochannel;(i) a circuit configured to control operation of the first, second,third and fourth electrodes to controllably thread, load and react DNAinto ordered fragments; and (j) an electrical or optical detector incommunication with the detection nanochannel configured to spatially andtemporally resolve fragment size to thereby allow an ordered restrictionmap of chromosomal DNA in real time or near real time.

The microfluidic channel can include an array of spaced apart postsconfigured to partially occlude the microfluidic flow path.

The system can include a nanofunnel connecting the microfluidic channelwith the ingress portion of the fluid transport nanochannel.

The nanofluidic reaction channel can have a serpentine shape with aplurality of closely spaced substantially parallel segments connected by“U” shaped segments.

The microfluidic channel, the reaction nanochannel, the detectionnanochannel and the first and second fluidic transverse channels can bemonolithically integrated on a fluidic chip.

The first and/or second transverse fluidic channel can be a fluidicnanochannel with depths between about 1 nm and about 100 nm and widthsbetween about 20 nm and about 2000 nm.

The reaction nanochannel can be between 10 and 1000 times longer andbetween 2 and 10 times larger in depth and/or width than the detectionnanochannel.

The microfluidic channel can be in fluid communication with one or morereservoirs, at least one of which includes whole cells for DNA analysis.

The whole cells for analysis can be restrained by a gel matrix for theextraction of genomic DNA.

The whole cells for analysis can be restrained by at least one highand/or low-density post array in the microfluidic channel to facilitateextraction of genomic DNA.

The microfluidic ingress channel includes whole cells with DNA. Thesecond transverse fluidic channel merges into a second fluid reservoirat an end portion away from the reaction nanochannel. The second fluidreservoir holds a solution of restriction endonuclease and cofactor.

The reaction nanochannel can be straight and can have a length betweenabout 500 μm to about 2 cm. The reaction nanochannel can be betweenabout 10 and about 1000 times longer and between about 2 and about 10times larger in depth and/or width than the detection nanochannel.

The system may include a second reaction nanochannel that is in fluidcommunication with the fluid microchannel on an ingress end of thesecond reaction nanochannel and that merges into a respective seconddetection nanochannel at an opposing egress end of the second reactionnanochannel.

Other embodiments are directed to nanofluidic analysis chips. The chipsinclude: (a) a microfluidic inlet adapted to extract genomic DNA fromwhole cells with a microfluidic channel having an array of posts; (b) areaction nanochannel that is between 500 μm and 10 cm long, the reactionnanochannel having an ingress portion that connects to the microfluidicinlet; (c) a detection nanochannel that merges with an egress end of thereaction nanochannel at an intersection defined by a reduction innanochannel size; (d) a first transverse nanochannel extending from andin fluid communication with the reaction nanochannel that is spacedapart from but proximate an ingress portion of the reaction nanochannel;and (e) a second transverse fluidic nanochannel extending from and influid communication with the reaction nanochannel downstream of thefirst transverse fluidic nanochannel.

The chip can also include a nanofunnel residing between and connectingthe reaction nanochannel with the microfluidic inlet.

The chip can include a plurality of reservoirs, including at least onein fluid communication with the microfluidic inlet, at least one influid communication with the first transverse nanochannel, at least onein fluid communication with the second transverse nanochannel, and atleast one in fluid communication with an end of the detectionnanochannel.

The array of posts in the microfluidic channel can include multiplesegments of arrays that are axially spaced apart.

The array of posts can be configured to extend across substantially anentire width of the microfluidic channel.

Still other embodiments are directed to methods of generating an orderedrestriction map of genomic DNA extracted from whole cells. The methodsinclude: (a) providing a device having a fluidic microchannel thatmerges into a reaction nanochannel that merges into a detectionnanochannel at an interface where the nanochannel diameter decreases insize; (b) lysing whole cells and dechromatinizing DNA with minimalfragmentation in the microchannel; then (c) introducing an intactmolecule of DNA to the reaction nanochannel; then (d) fragmenting theintact DNA in the reaction nanochannel using restriction endonucleaseenzymes. The reaction nanochannel is sized and configured so that thefragments stay in an original order until they are injected into thedetection nanochannel. The method further includes (e) detecting signalat one or more locations along the detection nanochannel to mapfragments in the order they occur along a long DNA molecule.

The device can include at least one reservoir in fluid communicationwith a fluidic microchannel that merges into the reaction nanochannel.The introducing step can be carried out by introducing whole cells foranalysis using the reservoir. The method can also include providing agel matrix and/or high-density post array for immobilizing the wholecells, then lysing the cells, extracting the DNA, and, optionally,staining the DNA, then introducing the intact molecule of DNA into thereaction nanochannel.

The device can include a microfluidic channel with an array of poststhat is in fluid communication with an ingress end of the reactionnanochannel and a first transverse channel in fluid communication withthe reaction channel downstream of the microfluidic channel.

The method can include threading and loading the sample by applying avoltage to the microfluidic and transverse channels to create a bias atthe ingress region of the reaction nanochannel.

The threading step can be carried out using controlled voltage orconcentration polarization gradients proximate the ingress of thereaction channel so that initially the DNA molecule is not subjected toa strain that exceeds DNA tensile strength and does not mechanicallybreak.

After the threading step, the loading can be carried out by changing thevoltages applied to the reaction nanochannel and transverse nanochannelsto pull the full DNA molecule into the reaction nanochannel at avelocity that is between about 1 μm/s and about 1 mm/s, such that atrailing end of the DNA molecule has sufficient time to disengagediffusively from any post entanglements and mechanical breakage isavoided.

The reaction of restriction digestion can be carried out by changing thevoltages applied to the reaction nanochannel and transverse nanochannelsto introduce restriction endonuclease and cofactor to the DNA containedin the reaction nanochannel, then a reaction is allowed to progressuntil all restriction sites have been digested.

The voltages applied to the reaction nanochannel and transversenanochannels can inhibit or prevent the introduction of restrictionendonuclease and cofactor to the microfluidic channel and thus preventthe digestion of DNA molecules external to the reaction nanochannel.

The detection of the ordered restriction fragments can be carried out bychanging the voltages applied to the reaction nanochannel and transversenanochannels to drive migration of the fragments to the interfacebetween the reaction nanochannel and the detection nanochannel.

The detection nanochannel diameter can decreases in size by between 50%to 99% from the reaction nanochannel thereby resulting in an increase intransport velocity as each fragment reaches the intersection and theseparation of each neighboring fragment. Then the detecting step can becarried out to detect transport of the separated fragments through thedetection nanochannel.

The detecting step can be carried out by detecting the fragmentsoptically or electrically at one or more locations along the detectionnanochannel.

The method can include determining fragment size by analyzing a detectedsignal duration or integrated amplitude.

The device can be a fluidic analysis chip.

The chip can be used in combination with a transport system that is incommunication with the chip, wherein the transport system is configuredto apply at least one of electrokinetic, pressure, or centripetal forcesto cause transport genomic DNA and fragments thereof through thereaction nanochannel into the detection nanochannel.

Yet other embodiments are directed to methods for interfacing withfluidic analysis chips. The methods include: (a) controlling sampleintroduction, cell lysis, and DNA extraction and staining; (b) DNAthreading and loading extracted DNA into the reaction nanochannel,restriction digestion with restriction endonuclease and cofactor in thereaction nanochannel, and an ordered transport of restriction fragmentsthrough the detection nanochannel; (c) electronically detectingrestriction fragments during transport through the detectionnanochannel; (d) electronically analyzing restriction fragment sizesfrom DNA molecules using real time or near real time analysis; and (e)electronically generating consensus restriction maps from multiple DNAmolecules and assessing map quality to evaluate the need for additionalsampling.

Still other aspects of the invention are directed to systems forinterfacing with fluidic analysis chips. The systems include: (a) meansfor controlling sample introduction, cell lysis, and DNA extraction andstaining; (b) means for controlling DNA threading and loading into thereaction nanochannel, restriction digestion with restrictionendonuclease and cofactor in the reaction nanochannel, and an orderedtransport of restriction fragments through the detection nanochannel;(c) means for detecting restriction fragments during transport throughthe detection nanochannel; (d) means for analyzing restriction fragmentsizes from DNA molecules using real time or near real time analysis; and(e) means for generating consensus restriction maps from multiple DNAmolecules and assessing map quality to evaluate the need for additionalsampling.

Still other embodiments are directed to fluidic analysis devices. Thedevices include a microfluidic channel having a flow path merging into areaction nanochannel; a DNA reservoir in fluid communication with themicrofluidic channel upstream of the reaction nanochannel; a threadingreservoir in fluid communication with the reaction nanochannel residingproximate the inlet of the reaction nanochannel; a detection nanochannelconnected to an egress end of the reaction nanochannel having a smallerdiameter and/or smaller width and depth than the reaction nanochannel;and a reservoir comprising restriction endonuclease and cofactor influid communication with the reaction nanochannel, residing proximatethe detection nanochannel.

It is noted that aspects of the invention described with respect to oneembodiment, may be incorporated in a different embodiment although notspecifically described relative thereto. That is, all embodiments and/orfeatures of any embodiment can be combined in any way and/orcombination. Applicant reserves the right to change any originally filedclaim and/or file any new claim accordingly, including the right to beable to amend any originally filed claim to depend from and/orincorporate any feature of any other claim or claims although notoriginally claimed in that manner. These and other objects and/oraspects of the present invention are explained in detail in thespecification set forth below. Further features, advantages and detailsof the present invention will be appreciated by those of ordinary skillin the art from a reading of the figures and the detailed description ofthe preferred embodiments that follow, such description being merelyillustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of a fluidic analysis deviceaccording to embodiments of the present invention.

FIG. 1B illustrates a cover plate can be bonded to the device shown inFIG. 1A according to embodiments of the present invention.

FIG. 1C illustrates that reservoirs may be attached to the device shownin FIG. 1B according to some embodiments of the present invention.

FIGS. 2A-2D illustrate an on-chip process of DNA extraction according toembodiments of the present invention.

FIGS. 3A-3C are schematic illustrations of an enlarged microfluidicportion of a device showing capturing of whole cells, lysis and stainingof dechromatinized DNA according to embodiments of the presentinvention.

FIG. 4A is a schematic illustration of a fluidic analysis device with anexemplary pattern of sample inlets that lead to the nanofluidic reactionchannel according to embodiments of the present invention.

FIG. 4B is a schematic illustration of a fluidic analysis device withmultiple reaction nanochannels and respective detection nanochannels influid communication with a common microchannel through which sample DNAmolecules are introduced according to some embodiments of the presentinvention.

FIG. 5 is a greatly enlarged “to scale” illustration of an interfacebetween a microfluidic channel and a nanofluidic reaction channelaccording to embodiments of the present invention.

FIGS. 6A-6D are schematic illustrations of a fluidic analysis devicewith exemplary voltage and operational sequences according toembodiments of the present invention.

FIG. 6E is an enlarged view of the detection nanochannel with a multiplepoint detection circuit (rather than the single point circuit of FIG.6D) according to embodiments of the present invention.

FIG. 6F is a schematic illustration of an analysis device similar to thedevice shown in FIGS. 6A-6E that employs pressure transport systemsinstead of voltage drive systems according to alternate embodiments ofthe present invention.

FIG. 7A-7D are schematic illustrations of a fluidic analysis device withexemplary voltage and operational sequences similar to FIGS. 6A-6D, butwith an alternate detection circuit according to embodiments of thepresent invention.

FIG. 7E is an enlarged view of the detection nanochannel with a multiplepoint detection circuit (rather than the single point circuit of FIG.7D) according to embodiments of the present invention.

FIG. 8 is an image of a prototype fluidic device fabricated using FIBmilling illustrating the fluidic structures (the enlarged insert is ofthe intersection between the reaction and detection nanochannelsaccording to embodiments of the present invention.

FIG. 9A is a bright field optical image of the intersection of thereaction nanochannel with the nanochannel for introducing Mg²⁺ accordingto embodiments of the present invention.

FIG. 9B is a difference image of the region of the device shown in FIG.9A showing the increase in Magnesium Green fluorescence uponvoltage-gated introduction of Mg²⁺ ions according to embodiments of thepresent invention.

FIG. 9C is a difference image of the region of the device shown in FIG.9A showing minimal change in Magnesium Green fluorescence when the opengate voltages were applied but no Mg²⁺ was present in the sidenanochannel according to embodiments of the present invention.

FIG. 10A is a series of fluorescence images showing the diffusion ofλ-DNA in a reaction nanochannel.

FIG. 10B is a series of fluorescence images showing that after about a1.5 minute digestion by a restriction endonuclease in the presence ofMg²⁺ three fragments are observable. Fragment order was retained despitethe fragments' high diffusivity.

FIG. 11 is an image of a series of frames showing a fluorescentlystained T4-phage DNA molecule fragmented in a 400 nm by 400 nm reactionnanochannel and injected into a 200 nm by 200 nm detection channelswhere the fragments are spatially well resolved during transport (lowertwo enlarged frames c and d) The middle inset shows an SEM image of theinterface between the reaction and detection nanochannel segments. Thearrow indicates time.

FIG. 12 is a schematic illustration of a system for restriction mappingof chromosomal DNA according to embodiments of the present invention.

FIG. 13A is a schematic illustration of the optical detection of DNAthreading into the reaction nanochannel.

FIG. 13B is a schematic illustration of the electrical detection of DNAthreading into the reaction nanochannel.

FIG. 13C is a representative voltage program for the threading, loading,reacting, and detecting of DNA molecules, with triggering initiated bythe threading detection circuits and fragment detection circuits.

FIG. 14 is a flow chart of exemplary operations that can be carried outto map DNA fragments according to embodiments of the present invention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein. Like numbers refer to like elementsthroughout. In the figures, certain layers, components or features maybe exaggerated for clarity, and broken lines illustrate optionalfeatures or operations unless specified otherwise. In addition, thesequence of operations (or steps) is not limited to the order presentedin the figures and/or claims unless specifically indicated otherwise. Inthe drawings, the thickness of lines, layers, features, componentsand/or regions may be exaggerated for clarity and broken linesillustrate optional features or operations, unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms, “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including” when used in thisspecification, specify the presence of stated features, regions, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, regions, steps,operations, elements, components, and/or groups thereof. As used herein,the term “and/or” includes any and all combinations of one or more ofthe associated listed items. As used herein, phrases such as “between Xand Y” and “between about X and Y” should be interpreted to include Xand Y. As used herein, phrases such as “between about X and Y” mean“between about X and about Y.” As used herein, phrases such as “fromabout X to Y” mean “from about X to about Y.”

It will be understood that when a feature, such as a layer, region orsubstrate, is referred to as being “on” another feature or element, itcan be directly on the other feature or element or intervening featuresand/or elements may also be present. In contrast, when an element isreferred to as being “directly on” another feature or element, there areno intervening elements present. It will also be understood that, when afeature or element is referred to as being “connected”, “attached” or“coupled” to another feature or element, it can be directly connected,attached or coupled to the other element or intervening elements may bepresent. In contrast, when a feature or element is referred to as being“directly connected”, “directly attached” or “directly coupled” toanother element, there are no intervening elements present. Althoughdescribed or shown with respect to one embodiment, the features sodescribed or shown can apply to other embodiments.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention.

The term “nanochannel” refers to a channel or trench having a criticaldimension that is at a nanometer scale. The nanochannel has sidewallsand a floor. The nanochannel can be formed into a solid substrate tohave an open top surface and a closed bottom surface with the sidewallsextending therebetween. A cover may be used to seal or otherwise closethe upper surface of the nanochannel(s). The term “primary dimension”refers to a width and/or depth dimension. The primary dimensions of afluid transport nanochannel can be between about 1 nm to about 500 nm.Different nanochannels can have different primary dimensions. Theprimary (also known as “critical”) dimensions of the reactionnanochannel can be between about 300-400 nm and the reaction nanochannelcan be between about 10 nm to about 300 nm.

The term “about” refers to parameters that can vary between +/−20% orless, such as +/−10%.

The term “transverse” nanochannel refers to a fluidic nanochannel thatcrosses a respective fluid transport nanochannel.

The term “fluid transport nanochannel” refers to a nanochanneltherethrough which an analyte flows for analysis. In certainembodiments, the fluid transport nanochannel can have two primarysegments, a reaction nanochannel and a detection nanochannel. Theanalyte can be any analyte of interest including, for example, singleanalyte molecules including synthetic and biological macromolecules,nanoparticles, small molecules, DNA, nucleic acids/polynucleic acids,peptides, proteins and the like. The transport through the nanochannelcan be carried out using electrokinetics, concentration polarizationand/or hydraulic pressure (forced pressure or pressure gradients).

The term “upstream” indicates a relative position that is closer to thefluid transport nanochannel or reaction channel ingress. The term“downstream” indicates a relative position that is closer to the fluidtransport nanochannel or reaction channel egress.

The term “shallow” refers to nanochannel depths that have a lesser depththan a transport nanochannel and that are smaller than analytemacromolecules' hydrodynamic sizes. With respect to the depth of thereaction nanochannel, a shallow nanochannel has a depth that istypically less by at least a factor of 2, such as by between 2-100×.Thus, for example, a shallow nanochannel segment can be 10 nm or less,typically between about 0.1 nm and 9 nm, while the transport nanochannelcan have a depth (at least adjacent the shallow segment) that is 20 nmor more, such as between 20-100 nm.

The term “long” with respect to the reaction nanochannel 20 means thatthe reaction nanochannel is between 10 and 1000 times the length of thedetection nanochannel 40. The reaction nanochannel 20 can be longer andbetween 2 and 10 times larger in depth and/or width than the detectionnanochannel 40. The reaction nanochannel 20 can, in some embodimentshave a length between about 500 μm and 10 cm long.

The term “wide” means that the nanochannel has a width that is at least2× (two times, “X” means “a multiplier” or “times”) that of a width ofthe transport nanochannel that it cooperates with to perform theanalysis (e.g., provide a driving voltage), and more typically between3×-100×, such as 3×, 4×, 5×, 6×, 7×, 8×, 9×, about 10×, about 20×, about40×, about 50×, about 60×, about 70×, about 80×, about 90×, or about100× the width of the adjacent cooperating reaction nanochannel.

The term “circuit” refers to an entirely hardware embodiment or anembodiment combining software and hardware.

The term “high density” with respect to the posts means that the arraysextend across the entire width of a microchannel and the posts arearranged with an edge-to-edge spacing that is less than about 10 μm. Theterm “low density” means that the posts are arranged with anedge-to-edge spacing that is typically greater than about 50 μm.

The term “low velocity” means that the macromolecule moves through thenanochannel at a velocity that is between about 1 μm/s and about 1 mm/s.

The term “significantly different field strengths” means that one sideof the fluid transport nanochannel can have a voltage/cm field strengththat is 10×-1000×, typically 100×-200×, greater or smaller than a secondsegment of that same channel.

The term “thread” and derivatives thereof means the process by which theanalyte molecule is initially introduced to the reaction nanochannel 20,providing for the linearization of a macromolecule from the random coilconformation realized in the microchannel or reservoir. The term “load”means that an analyte molecule present in a microchannel or reservoiraccessing the entrance(s) to the reaction nanochannel 20 is successfullyintroduced to the reaction nanochannel in its entirety and in a linear,post-thread configuration.

The term “react” and derivatives thereof means that DNA is fragmentedusing restriction endonuclease enzymes and an optional cofactor. Forexample, Mg²⁺ is a cofactor for a Type II class of restrictionendonucleases that may be particularly suitable for embodiments of thepresent invention. The fact that the cofactor is charged can aid involtage gating of the second transverse channel 32. The majority ofrestriction endonucleases that are available are Type II. Other types(Types I, III, IV) may also be suitable and have different cofactors(ATP, S-adenosyl-L-methionine) that may be controlled in a similarmanner. Thus, while preferred, embodiments of the invention are notlimited to Type II with a Mg²⁺ cofactor.

The term “size” means that fragments are pulled into the detectionnanochannel, creating separation from neighbors for the determination ofthe size of fragments by detecting electrical or optical signal durationor amplitude.

Embodiments of the invention are directed to genomic mapping of DNA in ananofluidic device.

FIGS. 1A-1C illustrate an exemplary nanofluidic analysis device 10. Inthis embodiment, the device 10 can be a chip with a pattern ofmicrochannels 15 and nanochannels 20, 30, 32 and 40. FIG. 1B illustratesa cover 11 that can be attached (typically bonded) to a substrate 12with the pattern of channels. FIG. 1C illustrates reservoirs 50 can beattached to the device 10. As shown, the device 10 includes amicrofluidic channel 15 that connects to an ingress end of the (long)reaction nanochannel 20. The detection nanochannel 40 is not required tobe inline with the reaction channel 20 and can angle away from orotherwise extend from the reaction nanochannel. The other end of thereaction channel 20 merges into a detection nanochannel 40 at aninterface I that is defined by a reduction in size, e.g., width/depthand/or diameter of the nanochannel. A first transverse channel 30extends off the reaction nanochannel 20 proximate the ingress end of thereaction nanochannel, typically within about 10-500 μm downstream of thenanofunnel 17 (where used) and/or ingress portion of the reactionnanochannel 15 e.

A second transverse channel 32 extends off the reaction nanochannel 20downstream of the first transverse channel 30 and before the interfaceI.

In some embodiments, the monolithic integration of a number ofnanofluidic components results in the rapid generation of genome levelmaps of DNA extracted from whole cells using the nanofluidic device 10.

Generally stated, a suspension of whole cells can be introduced to amicrofluidic input (one or more of the reservoirs 50) on the device 10.The cells are lysed and the DNA is dechromatinized and, in someembodiments, fluorescently stained. Intact chromosomal DNA is thenintroduced to a long reaction nanochannel 20, which extends the moleculeand prevents the diffusive mixing of fragments generated in thesubsequent steps. A solution of restriction endonuclease and cofactor isthen introduced to the reaction nanochannel 20, resulting in thedigestion of the DNA at sequence specific restriction sites. The lengthsof these fragments are then analyzed by transporting the orderedfragments contained in the reaction nanochannel 20 to the intersection Iwhere the reaction nanochannel 20 is interfaced to the detectionnanochannel 40. The force driving transport (e.g., electrostatic,pressure, or centripetal) is greater in the detection nanochannel 40than in the reaction nanochannel 20, resulting in an increase intransport velocity as each fragment reaches the intersection and theseparation of each fragment from its neighbors. The spatially andtemporally resolved fragments are detected downstream in the detectionnanochannel 40 using imaging or single point or multiple point detection(electrical or optical) and the resulting signal analyzed to determinethe fragment size. In this fashion, an ordered restriction map ofchromosomal DNA can be produced in real time or near real time. The term“near real time” means in a time that is within about 1 minute of realtime due to bandwidth of operational systems or other analysis-relatedcomputation or lag time.

In some embodiments described herein, device operations are primarilyelectrostatically controlled using voltages applied at the variousfluidic inlets but other forces (e.g., pressure or centripetal) can alsobe used as will be recognized by those of skill in the art.

Device Fabrication

Fluidic devices can be fabricated in a variety of substrates includingsilicon, glass (silica), quartz, plastics, thermoplastics, andelastomers or a combination thereof. FIGS. 1A-1C show an example devicefabrication workflow. Microfluidic components 25 shown by thelarger/wider/darker lines that facilitate DNA extraction and provide aninterface to the device's nanofluidic elements can be patterned usingestablished methods such as photolithography and wet or dry etching,molding, embossing, or machining. The nanofluidic elements 20, 30, 32,40, can be fabricated using a variety of methods includingphotolithography, electron beam lithography, or nanoimprint lithographyfollowed by etching; focused ion beam milling; electron beam milling;molding; or embossing. Once the fluidic elements are fabricated in thetop surface of the substrate 12, a cover plate 11 can be attached,typically bonded to the substrate to form the enclosed fluidic networkusing, for example, fusion bonding, anodic bonding, or bonding with anadhesive film between the bottom substrate and cover plate. Themicrochannels can be accessed through vias that pass through the bottomsubstrate and/or top cover plate. Reservoirs 50 can be affixed to thedevice over the vias 50 v to facilitate liquid handling. Electrodes 50 ecan be inserted into all or selected reservoirs 50. The reservoirs 50have vias 50 v. The electrodes 50 e apply voltages across the variousfluidic elements. Air or vacuum lines can be coupled to the reservoirsor vias to apply positive pressure or vacuum to the fluidic elements anddrive pressure-driven fluid flow.

DNA Extraction

The encapsulation of cells in gelling media before or during theirintroduction to a fluidic device, or their capture in a network ofnanometer or micrometer-scale fabricated structures enables theextraction of chromosomal DNA from the cells with little or nofragmentation. As an example, the use of low melting point agarose gelfor the manipulation of cells is shown in FIG. 2A-2D. Cells in lowmelting point agarose are introduced to a microfluidic reservoir (FIG.2A) and then pulled into the inlet microfluidic channel while theagarose is still melted, using a syringe pump withdrawing from thechannel outlet (FIG. 2B). The agarose is allowed to gel, encapsulatingand protecting the DNA during subsequent treatments. Solutions areintroduced to digest the cell wall (for microbes and plant cells) thento chemically lyse the cell using detergent solutions incorporatingagents such as proteinase K to inhibit native nuclease activity (FIG.2C). The gel is rinsed thoroughly with buffer, followed by a solution ofintercalating dye. Incorporating these tasks on a chip enables theprecise control of flow rates and eliminates any turbulence that mightotherwise contribute to DNA shearing. The gel is melted by heating thedevice (it may be further disrupted by adding the enzyme agarase) andthe dechromatinized DNA extracted from the matrix electrophoretically(FIG. 2D). The uncharged agarose is excluded from the nanofluidic regionof the device by electroosmotic flow. Restriction endonuclease can beadded to the DNA before it encounters the nanofluidic channels through aseparate inlet microchannel (not shown).

Alternative approaches to the agarose encapsulation indicated hereinclude using a microfabricated high density post array 16 to trap cellsintroduced to the device using pressure-driven flow, lysing the cells,and then capturing the DNA by entanglement within the post array (FIGS.3A-3C). FIG. 3A shows capture of whole cells. FIG. 3B shows lysis ofcells and staining of dechromatinized DNA using low flow rates and/ordiffusive mixing of reagents. FIG. 3C illustrates extraction of the DNAfrom the post array 16 using an applied voltage. The posts 16 can becircular or have other geometries, typically without sharp edges. Theposts 16 can have the same height as the depth of the fluidicmicrochannel 15. The posts can have small spaces therebetween to allowfor an uncoiled length of the DNA to travel therebetween. In someembodiments, the posts 16 can have a width of between about 1-10 μm,typically about 5 μm with spacing between posts greater or lesser thanthe width of the posts, typically between about 2-50 μm, such as about10 μm.

A pattern of multiple fluid inputs 15 a of microfluidic channels 15 cand reservoirs 50 can also be used for sample introduction and DNAextraction for analyses requiring more material (FIG. 4A).

FIG. 4B illustrates that the device 10 can include a plurality ofreaction nanochannels fed DNA from a single microchannel 15 to increaseprocessing speeds. Although shown as two reaction nanochannels 20 ₁, 20₂ merging into respective detection nanochannels 40 ₁, 40 ₂ and incommunication with respective transverse channels 30 ₁, 30 ₂, 32 ₁, 32₂, more than two transport nanochannels and associated components may beused, e.g., between 2-100 on a single chip, for example.

Introducing Long Genomic DNA Molecules to the Reaction Nanochannel

In order to overcome an entropy-based energy barrier to DNA confinement,significant forces can be imposed on large DNA molecules in order tointroduce them to a nanochannel. Strategies to facilitate DNA threadinginto the reaction nanochannel 20 without shearing include theincorporation of gradient structures and/or means of quickly reducingthe field strength after threading is initiated in order to reducestress on the molecule. As one example, using focused ion beam (FIB)milling, structures with gradually decreasing width and depth(nanofunnels) can be fabricated to serve as conduits for DNAintroduction to a seamlessly interfaced nanochannel. Furtherdescriptions of nanofunnels can be found in U.S. Provisional ApplicationSer. No. 61/597,364 and PCT/US2013/025078, the contents of which arehereby incorporated by reference as if recited in full herein. Inanother example, intersecting nanofluidic elements can be used to gaingreater control of DNA transport as described in U.S. Provisional PatentApplication Ser. No. 61/770,586, the contents of which are herebyincorporated by reference as if recited in full herein. FIG. 5 shows theincorporation of both of these technologies in a single device, togetherwith a low-density array of posts 16 that encourages DNA linearization.These posts 16 can be provided as a plurality of axially spaced apartarray segments or groups of posts 16 ₁-16 ₄, although more or fewersegments of the same or different post array size and/or configurationcan be used to partially occlude the travel path and force the DNA totravel between adjacent posts. The axial distance or spacing betweenpost segments can be between 20-200 μm, typically about 100 μm. Thelength of the high-field section F_(H) of the nanochannel 20 shown inFIG. 5 determines the force on the molecule and is relatively short toprevent undue stress on the DNA. Similarly, the distance between thelast row of posts and the nanochannel entrance is great enough that theinitial threading of DNA can occur without pulling the DNA taut. Loadingthe full DNA molecule occurs at a low velocity so that the trailing endof the molecule has sufficient time to disengage diffusively from anypost entanglements. The field strength should be high enough, however,to counter the diffusion and entropic recoil that favor de-threading.The microfluidic/nanofluidic interface shown in FIG. 5 is merely oneexample of many structures that can be engineered for the controlledintroduction of DNA to the reaction nanochannel.

Restriction Fragmentation and Fragment Sizing

FIGS. 6A-6E show exemplary analysis steps conducted in the device's 10nanofluidic channels 20, 30, 32, 40 that lead to the generation of anordered restriction map, beginning with the threading and loading ofgenomic DNA described above (FIGS. 6A-6B). This is followed by therestriction digestion of the extended DNA molecule into fragments (FIG.6C). The restriction endonuclease enzymes that digest the DNA moleculeare contained in the bottom microchannel interfaced to the secondtransverse channel 32 in FIG. 6C (labeled by way of example only as“Mg²⁺”) and can also be introduced with the DNA through the entrance tothe reaction nanochannel shown on the left hand side of FIG. 6C.Restriction endonucleases that require a cofactor (e.g., Mg²⁺ ions) canbe used to ensure that DNA fragmentation occurs only with equilibratedDNA molecules that are fully confined in the reaction nanochannel. Thisis accomplished by the controlled introduction of the cofactor at theappropriate time to affect restriction digestion, most easily by theapplication of appropriate voltages in the four channel inlets V0, V1,V2, V3 shown in FIGS. 6A-6E and 7A-7E. The polarity and magnitude of theelectric fields in each of the channels during each mode of operationare indicated by arrows in FIGS. 6A-6E. Since, in this example, thecofactor is a positively charged ion, it can be electrophoreticallyexcluded from or introduced to the reaction nanochannel 20. FIG. 6Dshows the injection of DNA fragments into the detection nanochannel 40with the aid of an array of posts 16 and a nanofunnel 17. In thisexample, the effective diameter of the detection nanochannel 40 issmaller than that of the reaction nanochannel 20. The reactionnanochannel 20 can be between 10 and 1000 times longer and between 2 and100 times larger in depth and/or width than the detection nanochannel40. For example, the reaction nanochannel 20 can have a width and depthof about 300 nm while the detection nanochannel can have a smaller widthand depth, e.g., a width and depth of about 100 nm. In some embodiments,the detection nanochannel 40 has a diameter that decreases in size bybetween 50% to 99% from that of the reaction nanochannel 20 therebyresulting in an increase in transport velocity as each fragment reachesthe intersection and the separation of each neighboring fragment.

As a result of this channel constriction, the electric field in thedetection nanochannel 40 is greater than that in the reactionnanochannel 20 and DNA restriction fragments are rapidly pulled into thedetection nanochannel when they arrive at the intersection. There is aninter-fragment period of time before the next fragment migrates to theintersection and is pulled into the detection nanochannel 40. As thefragments translocate through the detection nanochannel 40, they aredetected downstream and the signal duration or integrated intensity isanalyzed to determine the fragment size. In FIG. 6A-6D, this isaccomplished by a circuit 100 for detecting the fluorescence of stainedDNA fragments passing through a focused laser spot using an avalanchephotodiode 102 (FIG. 6D) or multiple point detection (FIG. 6E) using,for example, two lasers 103 through a single objective lens (Laser 1 andLaser 2) or using additional objective lenses (Laser 3). Detection canalso be achieved using a circuit 100′ for fluorescence imaging.

In FIGS. 7A-7E, electrical single point (FIG. 7D) and multiple point(FIG. 7E) detection is illustrated (e.g., using an opposed pair ofelectrodes integrated with the detection nanochannel and an ammeter 101to detect fragment-induced changes in conductance).

FIG. 6F illustrates the device 10 can be configured to operate using apressure driven transport system of pressure/vacuum 50 p via reservoirs50 to cause the DNA molecule and fragments to thread, load and/ortransport according to alternate embodiments of the present invention.The device can include conduits or tubes that connect to the pressuresources and allow automated operation along the lines noted for theelectrokinetic or voltage systems. The operation of the device shown inFIG. 7A and as shown for figures illustrating other embodiments can alsobe operated with a pressure or other transport system. Thus, the device10 can operate with different transport systems, such as at least one ofelectrokinetic, pressure, or centripetal forces that can be applied tocause transport of genomic DNA and fragments thereof through thereaction nanochannel into the detection nanochannel.

The interface between the reaction nanochannel and detection nanochannelshown in FIGS. 6A-6E and 7A-7E should also be considered one example ofa class of structures where an abrupt change in the forces drivingfragment transport results in fragment spatial and/or temporalresolution.

The reaction nanochannel 20 shown in FIGS. 6A-6D and 7A-7D isillustrated as having a serpentine shape with multiple parallel legsconnected by “U” shaped segments. However, other nanochannel shapes canbe used including straight lengths for shorter channels typicallybetween about 500 μm to 2 cm. The serpentine shape may be particularlysuitable for longer channels on a monolithic chip.

The elements of restriction digestion, fragment resolution throughinjection into a detection nanochannel, and fragment sizing illustratedin FIGS. 6C-6D have been demonstrated on prototype devices (FIG. 8).These prototype devices were fabricated using focused ion beam millingand have relatively short reaction nanochannels suitable for analysis ofviral or bacterial chromosomal DNA.

An advantage of an integrated nanofluidic network over nanochannels witha single input and a single output is the ability to control fluid flowsusing voltages at a variety of locations. In the digestion of DNA withrestriction endonucleases, it can be important to control theconcentration and location of the cofactor (e.g., Mg²⁺ ions) to ensuredigestion of the confined DNA while preventing the digestion of DNA yetto be introduced to the reaction nanochannel. In order to demonstratethis capability, a Mg²⁺ sensitive dye (Magnesium Green) inelectrophoresis buffer was introduced to the nanofluidic reaction anddetection channels of a prototype device. Buffer with magnesium chloride(10 mM) was added to the bottom channel, as indicated in FIG. 9. The“magnesium gate” was switched between closed and open states by applyinga small negative or positive voltage to the Mg²⁺ reservoir,respectively, while the reaction nanochannel was held at ground.Fluorescence images were collected using a 2-s exposure with themagnesium gate closed followed by an image when the gate was opened. Theintensity of the Magnesium Green increased as Mg²⁺ ions migrated downthe reaction nanochannel. FIG. 9B shows the increase in fluorescenceduring the experiment. This panel was generated by subtracting aninitial frame of the recorded series (collected when the magnesium gatewas closed) from the final frame with the gate open. To ensure that theincrease in fluorescence was not due to concentration polarization ofthe dye—a phenomenon observed in nanofluidic experiments—a controlexperiment was conducted in which the buffer in the bottom channel wasfree of Mg²⁺ ions. FIG. 9C shows that only a slight change in thefluorescence intensity was observed when the voltages were switched tothe “open” condition. It was also possible to direct the Mg²⁺ flow intothe top “threading reservoir” at the other end of the reaction channelthrough the first transverse channel 30, preventing its introduction tothe microchannel that serves as the source DNA reservoir in mappingexperiments.

The digestion of DNA molecules with a restriction endonuclease in thereaction nanochannel of a prototype device has also been demonstrated.Lambda phage DNA (λ-DNA) was stained with the intercalating dye YOYO-1(5:1 base pairs:dye molecule) in a buffer suitable for digestion usingthe restriction endonuclease HindIII. The buffer also contained EDTA (2mM) to sequester any Mg²⁺ that might poison the DNA-containingmicrochannels and mercaptoethanol (4% by volume) as a radical scavenger.HindIII was then added to the DNA solution and this solution loaded intothe DNA reservoir accessing the reaction nanochannel entrance. A secondsolution containing the reaction buffer without EDTA but with 10 mMmagnesium chloride, 4% mercaptoethanol, and HindIII was added to theMg²⁺ reservoir. The remaining reservoirs (labeled “Threading” and“Outlet” in FIG. 8) were filled with buffer containing 4%mercaptoethanol (i.e., no Mg²⁺, EDTA, or HindIII). Platinum electrodeswere immersed in the reservoirs, enabling independent control of thevoltages at the four inlets seen in FIG. 8. DNA molecules wereintroduced to the reaction nanochannel using a high field strength whilethe Mg²⁺ voltage gate was closed. When a λ-DNA molecule entered themicroscope's field of view, the voltages were switched to lower valuesso that DNA migration slowed and the Mg²⁺ voltage gate was opened,driving Mg²⁺ ions past the DNA molecule. After a few seconds, thevoltages were adjusted so that the field strength in the reactionnanochannel was approximately zero. Images were acquired every 200 ms.After an initial imaging period of 10-15 s, the shutter of thefluorescence excitation source was closed to ensure that nophoto-induced fragmentation occurred. After ˜1 min of reaction, theshutter was opened to determine the extent of digestion. FIG. 10 shows arepresentative series of frames indicating the digestion of λ-DNA aftera 1.5 min exposure to Mg²⁺. In this case, three fragments were observed,indicating the partial digestion of the DNA molecule by HindIII.

The resolution of DNA fragments from their neighbors in space and timethrough their ordered injection into a detection nanochannel has alsobeen demonstrated in a prototype device. Fluorescently stained T4-phageDNA molecules were injected into a nanochannel with dimensions (width xdepth) that were reduced from 400 nm×400 nm to 200 nm×200 nm. Given theequivalent length of the two segments, this corresponded to a four-foldincrease in the electric field in the smaller nanochannel. FIG. 11 showsthe diffusion of multiple fragments of a single phage molecule in thelarger reaction nanochannel. (For this relatively small DNA molecule,the fragments can be resolved over the observation period represented inthe figure. However, resolution of multiple large restriction fragmentsover larger fields of view would require extended observation periodsand be subject to greater uncertainties.) This observation period wasfollowed by the injection of fragments into the detection channel. Thisdynamic process effectively resulted in the full resolution of eachfragment from its neighbors.

Generation of Genome Level Restriction Maps

FIG. 12 shows a system 200 providing an example of how the variousdevice operations could be conducted using a bench-top apparatus 150with computer control 90 that can serially or concurrently test alow-cost, single-use device(s) 10. Typically, after a sample of wholecells is introduced to the device 10, it is inserted into the apparatus150. However, the device 10 may be loaded with cells after being placedin the apparatus 150. A timed program can be initiated in which reagentsare flowed past the cells to achieve DNA extraction and staining in thedevice 10. A predefined voltage program 90 p is initiated to thread thefirst piece of chromosomal DNA. The presence of threaded DNA (detectedoptically or electrically) triggers a change in the voltage program inorder to fully load the DNA molecule into the reaction nanochannel 20.When this has been achieved (as determined using optical or electricaldetection), the voltage program 90 p is again changed to initiate theendonuclease digestion reaction. During this step, the device 10 may beheated in the apparatus 150 to enhance reaction kinetics. Following aprescribed reaction time, the voltages are automatically changed to thevalues used to drive the ordered injection of fragments into thedetection nanochannel 40. The measured signal can be analyzed as it iscollected via circuit 100, 100′ (FIGS. 6D, 6E, 7D, 7E), which generatesa map of the restriction sites in real time. When the detection offragments ceases, the circuit 100, 100′ can indicate to the computer 90that the entire DNA molecule has been sampled and the “read” iscomplete. The voltages are changed to their “threading” values via thevoltage program 90 p to read the next piece of chromosomal DNA.

FIGS. 13A-13B illustrate exemplary elements and circuits 105, 105′ thatcan be used to detect the threading of a DNA molecule and FIG. 13C showsa timing/voltage control program with the triggering of the voltageprogram 90 p directed by input from one or more control circuits such as100, 100′, 105 and/or 105′. Triggered changes in the voltage program areindicated by vertical arrows. Delays (e.g., to provide for enough timeto complete the restriction digestion reaction) are indicated byhorizontal arrows. The process can be repeated many times to readmultiple copies of the chromosomal DNA that originated from the multiplecells initially introduced to the device. At the completion of eachread, that read can be computationally determined to be unique (i.e., itis the first time that chromosome has been mapped during theexperimental run) or aligned to existing map data or “reads” 160 toincrease read coverage (i.e., other copies of the chromosome have beenread previously in the experimental run). The long read lengths producedby this technology allow this process to be completed during the mappingexperiment. Consequently, a continuously updated quality score can beproduced and the analysis can be terminated after user-definedbenchmarks of map coverage and quality are achieved. Alternatively,restriction mapping using additional restriction endonucleases can beinitiated to generate high information content maps of variousrestriction sites.

FIG. 12 illustrates an automated analysis system 200 with a controlledenvironment housing 150 that can include a power source 150 p, fluidicconnections, and various chemicals for the reservoirs 50 (e.g., lysisreagents, rinse buffers, dechromatinization reagents, DNA stainingsolutions (where desired)). The system can control operations byinjecting and withdrawing fluids through the fluidic connections and byelectrically applying electrical biases, e.g., voltages, to electrodesin communication with the channels 20, 30, 32 and 40 (e.g., V0, V1, V2,V3 in FIGS. 6A-6D and FIGS. 7A-7D). The microfluidic channel 15, thefluid cross channels 30, 32 and the detection nanochannel 40 can mergeinto a reservoir 50 that is configured to hold a flowable substance suchas a fluid (electrolyte). The reservoir fluid can comprise anelectrolyte solution, e.g., a high ionic strength electrolyte solution.Examples of suitable solutions include, but are not limited to,potassium chloride solutions in concentrations from about 35 mM to about1 M.

Referring to FIG. 12, the system 200 can include voltage inputs 251-254to electrodes 50 e (FIG. 1C) for controllably applying V0, V1, V2, V3.The system 200 can include a power source 150 p (e.g., a voltage sourceand/or current source) that can apply the electrical bias underdirection of at least one processor 90 p with a desired voltage timingprogram or algorithm 90 p with a circuit 90 c that communicates with orincludes the detection circuit 100, 100′ (FIGS. 6D, 6E, 7D, 7E) and thethreading detection circuit 105, 105′ (FIGS. 13A, 13B). The system 200can apply and control voltages V0, V1, V2, V3 at the appropriate time tothread, load, react and transport and detect the molecule underanalysis. Alternatively, some functions can be achieved using pressuredriven flow by injecting or withdrawing solutions through fluidicconnections to the device 10 according to a timing program or algorithm90 p.

FIGS. 7A-7D illustrates a detection system using circuit 100′ electricaltriggering of voltage change and measuring transverse conductance usingan ammeter 101 and FIGS. 6A-6D illustrate a detection system 100 usingan avalanche photodiode 102 and laser 103 and electrical triggering ofvoltage change.

FIG. 12 shows the system 200 can include a computer 90 with a circuitand/or at least one processor 90 p that can obtain the analysis data forthe DNA fragments in the detection nanochannel 40. The term “computer”is used broadly to include any electronic device, typically comprisingat least one digital signal processor, allowing for control andcommunication with the circuit 100, 100′ and/or device 150 to controloperation. The computer can be local or remote from a site with thedevice 150.

The system can include an imaging system with a detector 102 andexcitation source 103 (FIG. 6D) that can take a series of images of ananalyte molecule in the detection channel 40. The imaging system can beany suitable imaging system. As shown, the system 100 can include anexcitation light source 103 (typically for generating light that excitesfluorescently labeled molecules) (which can optionally include a mirror,beam splitter, polarizer, lens, and/or other optical elements) and imagegenerating device or detector 102 such as one or more of a camera,photomultiplier tube or photodiode. The objective/lens, where used, canreside under or over a primary surface of the device 10. The electricinputs/outputs and flow operation can reside on an opposing side of thedevice 10. The device 10 may also be flipped to operate on its side(with the flat primary surfaces being upright or angled) rather thansubstantially horizontal.

FIG. 14 illustrates exemplary operations that can be used to provide DNAordered restriction maps of genomic DNA extracted from whole cellsaccording to embodiments of the present invention. A device having afluidic microchannel that merges into reaction nanochannel that mergesinto a detection nanochannel at an interface where the nanochanneldiameter decreases in size is provided (block 300). Optionally, the sizereduction can be between 50% to 99% from the size of the reactionnanochannel (block 302). Whole cells can be lysed and dechromatinized toproduce DNA with minimal fragmentation in the microchannel (block 310).Then an intact molecule of DNA can be introduced to the reactionnanochannel (block 315). Then the intact DNA can be fragmented in thereaction nanochannel using restriction endonuclease enzymes. Thereaction nanochannel is sized and configured so that the fragments stayin an original order until they are injected into the detectionnanochannel (block 320). Signal at one or more locations along thedetection nanochannel can be detected to map fragments in the order theyoccur along a long DNA molecule (block 325).

The device can be used with a transport system that is in communicationwith the device, so that the transport system is configured to apply atleast one of electrokinetic, pressure, or centripetal forces to causetransport of genomic DNA and fragments thereof through the reactionnanochannel into the detection nanochannel (block 321).

This technology allows for the controlled introduction of DNA from fullchromosomes to a nanochannel, its digestion with restriction enzymes,and the ordered mapping of restriction fragments. Injection-basedseparation of fragments to resolve neighboring fragments can minimizethe loss of resolution due to diffusion and reduce or eliminate thereliance on nanochannels having critical dimensions (width and depth)that approach or exceed the current limits of nanofabrication methods.

Advantageously, the smallest required nanochannel widths are typicallyabout 100 nm. Devices can therefore be fabricated using a variety ofroutine methods in various substrates. See, e.g., Mijatovic, D.; Eijkel,J. C. T.; van den Berg, A. Technologies for nanofluidic systems:top-down vs. bottom-up—a review. Lab Chip 2005, 5, 492-500; Perry, J.L.; Kandlikar, S. G. Review of fabrication of nanochannels for singlephase liquid flow. Microfluid. Nanofluid. 2006, 2, 185-193; Chantiwas,R. et al. Flexible fabrication and applications of polymer nanochannelsand nanoslits. Chem. Soc. Rev. 2011, 40, 3677-3702; and Utko, P.; andPersson, F.; Kristensen, A.; Larson, N. B. Injection molded nanofluidicchips: Fabrication method and functional tests using single-molecule DNAexperiments. Lab Chip 2011, 11, 303-308. The contents of which arehereby incorporated by reference as if recited in full herein. Theability to use wafer-scale processing can provide for a high impact, lowcost technology.

Chromosomal DNA can be extracted from cells on chip and introducedwithout intermolecular entanglements to a nanochannel for restrictiondigestion and fragment sizing. This ensures minimal DNA shearing,reducing the need for assembly of optical maps from many smalloverlapping contigs (contiguous consensus regions of DNA). This isexpected to increase throughput, reduce computational costs, and enablehigh coverage maps with low input material requirements.

Fragment sizes can be measured by imaging or single-point detectionusing the duration or integrated amplitude of the signal. DNA velocitycan be length independent for these measurements, which is expectedtheoretically and has been verified experimentally in channels of thissize. Data analysis can proceed in real time or near real-time, ensuringthat data can be collected in a single run until the desired coverageand map quality are achieved. The elimination of large field-of-viewimage storage and analysis can reduce computational costs.

Integration of additional functionality is possible. For example,selected fragments can be sorted after detection for further analysis.DNA could be subjected both to restriction digestion and to a secondassay such as a reaction with labeled methyl-CpG binding domain proteinsor peptides. See, e.g., Lim, S. F.; Karpusenko, A.; Sakon, J. J.; Hook,J. A.; Lamar, T. A.; Riehn, R. DNA methylation profiling innanochannels. Biomicrofluidics 2011, 5, 034106, the contents of whichare hereby incorporated by reference as if recited in full herein.Two-color detection could thus provide single-molecule epigeneticanalysis with sequence context.

Embodiments of the invention have potential for high impact primarily inthe areas of structural variant genotyping and de novo sequenceassembly. At present, the available genetic tests that assess forelevated disease susceptibility generally identify rare, high effectsingle nucleotide polymorphisms (SNPs) that are typically monogeniccoding errors. SNPs are not the only variants that are pathogenic,however, and genetic assessments would benefit from the inclusion ofstructural variants (novel insertions, deletions, duplications,inversions, and translocations). The contribution of structural variants(SVs) to disease phenotypes is less well understood than that of SNPs.Known examples include the association between SVs and schizophrenia,autism, and Crohn's disease. High-throughput, low-cost methods that canidentify SVs are therefore important complements to SNP-basedassociation studies. Current methods for identifying SVs (e.g.,hybridization-based array methods and computational methods foranalyzing next generation sequencing data) exhibit biases in the sizeand classes of variants detected, preventing global discovery. Inaddition to the biases inherent to each method, a large gap exists inthe detection of SVs between ˜300 and ˜10,000 base pairs.High-resolution restriction maps of chromosomal DNA provide astraightforward way to identify all classes of SVs present in anindividual's genome.

In addition to their utility in detecting SVs, optical maps can alsoserve as scaffolds for assembly of next generation sequencing contigs.See, e.g., Lam et al, Genome mapping on nanochannel arrays forstructural variation analysis and sequence assembly. Nat. Biotech. 2012,30, 771-776; Zhou et al., Whole-genome shotgun optical mapping ofRhodobacter sphaeroides strain 2.4.1 and its use for whole-genomeshotgun sequence assembly. Genome Res. 2003, 13, 2142-2151; and Zhou etal, A whole-genome shotgun optical map of Yersinia pestis strain KIM.Appl. Environ. Microbiol. 2002, 68, 6321-6331. The contents of which arehereby incorporated by reference as if recited in full herein.

Strategies that increase the throughput and decrease the cost ofrestriction site mapping can be of significant value for comparativegenomics studies. Additionally, the ability of restriction mapping tospan large highly repetitive regions will be valuable for assisting withdifficult assemblies such as heterochromatic DNA and plant genomes.

Embodiments of the Invention can accurately map fragments in the orderthat they occur along a large DNA molecule. This is facilitated by thenanochannel structure where the channel diameter decreases significantlyat the detection nanochannel (FIG. 11). In FIG. 11, this is shown by theseries of frames on the left hand side of the image. While in someframes at the top of the series, it is apparent that there are 4fragments, in other frames that is not obvious. Thus, one can obtain anestimate of the fragment sizes but it is not precise. Compare that tothe frames in the bottom two thirds of the series where there issignificant separation between fragments and greater precision indetermining the fragment sizes.

Embodiments of the invention are also configured to introduce a train offragments to the detection structure in the same order that they occurin the DNA molecule. To achieve this, an intact stretch of DNA can beintroduced to the reaction nanochannel 20 and then fragmenting the DNAwithin that reaction nanochannel using restriction endonuclease enzymes.These enzymes fragment the DNA only at sites that have a specificsequence (e.g., the HindIII enzyme recognizes the base sequence AAGCTTand cuts the DNA between the two A′s) generating a map of these sitesalong a molecule of DNA. The reaction nanochannel has a small enoughdiameter that the fragments do not intermix—they stay in the originalorder until they are injected into the detection nanochannel 40.

The above could be particularly suitable for introducing DNA moleculesthat are long, e.g., about 0.5 million base pairs long. It iscontemplated that if intact DNA that is 250 million base pairs long(i.e., an entire human chromosome's worth of DNA) can be introduced tothe reaction nanochannel 20, then this would greatly reduce analysistime, sample needed, and mapping errors. However, embodiments of theinvention can be beneficial for other uses such as a high impactdiagnostic and research tool.

While FIB milling is described for completeness and is believed to beparticularly suitable for forming the nanochannels, other embodimentsare directed to other forming techniques, as described above, including,for example, electron beam lithography, nanoimprint lithography,photolithography, templating or molding strategies, and other methodsunderstood by one of ordinary skill in the art.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

That which is claimed:
 1. A fluidic analysis method, comprising:providing a fluidic device, wherein the fluidic device comprises areaction nanochannel that is between 500 mm and 10 cm long and thatmerges into a detection nanochannel at an interface positiontherebetween where the detection nanochannel reduces in size relative tothe reaction nanochannel, wherein the reaction nanochannel has width anddepth dimensions that are between 1 nm and 500 nm, and wherein thereaction nanochannel is between 2 and 10 times larger in depth and/orwidth than the detection nanochannel, the fluidic device furthercomprising a fluidic microchannel in communication with an ingressportion of the reaction nanochannel, a first transverse fluidic channelextending from and in fluid communication with the reaction nanochannelat a location that is spaced apart from but proximate the ingressportion of the reaction nanochannel, and a second transverse fluidicchannel extending from and in fluid communication with the reactionnanochannel downstream of the first transverse fluidic channel, whereinthe first and second transverse fluidic channels have a depth that isless than the depth of the reaction nanochannel; introducing a DNAmolecule into the reaction nanochannel from the microfluidic channel;introducing restriction endonuclease enzyme or enzymes into the reactionnanochannel; introducing enzyme cofactors into the reaction nanochannel;fragmenting the DNA molecule by a restriction digestion reaction usingthe restriction endonuclease enzyme or enzymes in the reactionnanochannel until all restriction sites have been digested only in thereaction nanochannel, maintaining an original order in which they occuralong the DNA molecule; then injecting the ordered fragments in theiroriginal order into the detection nanochannel from the reactionnanochannel with neighboring fragments having increased spatialseparation relative to their separation in the reaction nanochannelproximate the interface position; and detecting signal for eachseparated fragment to determine fragment size to thereby allow anordered restriction map of DNA.
 2. The method of claim 1, furthercomprising generating an ordered restriction map in real time or nearreal time using the detected signal.
 3. The method of claim 1, whereinthe injecting step comprises increasing transport velocity of fragmentsat the interface position to spatially separate the neighboringfragments.
 4. The method of claim 1, further comprising controllablyapplying voltages to a first electrode associated with the fluidicmicrochannel, a second electrode associated with the first transversefluidic channel, a third electrode associated with the second transversefluidic channel and a fourth electrode associated with the detectionnanochannel to carry out the introducing steps and the fragmenting andinjecting steps.
 5. The method of claim 1, wherein the introducingenzyme cofactors comprises introducing enzyme cofactors into the secondtransverse fluidic channel, and wherein the DNA molecule is a long DNAmolecule having at least 0.5 million base pairs.
 6. The method of claim1, further comprising controllably changing polarity and magnitude ofelectric fields using electrodes associated with the microfluidicchannel, the reaction nanochannel, the detection nanochannel and thefirst and second transverse fluidic channels to carry out theintroducing, fragmenting and injecting steps.
 7. The method of claim 1,further comprising controllably applying at least one of electrokinetic,pressure, or centripetal forces to cause transport of the DNA moleculeand fragments thereof through the reaction nanochannel into thedetection nanochannel.
 8. The method of claim 1, further comprisingcontrollably applying at least one of electrokinetic, pressure, orcentripetal forces to the microfluidic channel, the reactionnanochannel, the detection nanochannel and the first and secondtransverse fluidic channels to carry out the introducing, fragmentingand injecting steps.
 9. The method of claim 1, wherein the fluidicdevice comprises multiples of the reaction nanochannel, the detectionnanochannel and the first and second transverse fluid channels, andwherein the method comprises concurrently introducing, fragmenting andseparating a plurality of DNA molecules in respective sets of thereaction and detection nanochannels.
 10. The method of claim 1, whereinthe DNA molecule is a long DNA molecule comprising at least 0.5 millionbase pairs.